- Email: [email protected]
A diallel analysis of the genetic underpinnings of mouse sleep
Physiology and Behavior, Vol. 12, pp. 169-175. Brain Research Publications Inc., 1974. Printed in the U.S.A.
A Diallel Analysis of the Genetic Underpinnings of Mouse S l e e p I JOYCE K. FRIEDMANN 2
University o f Florida, Gainesville, Florida, U.S.A.
(Received 15 March 1972) FRIEDMANN, J. K. A Diallel analysis of the genetic underpinnings of mouse sleep. PHYSIOL. BEHAV. 12(2) 169-175, 1974.-Four highly inbred mouse strains were crossbred in all possible combinations. Five young adult males in each of the four original and twelve offspring groups were chronically implanted with cortical, hippocampal and nuchal electrodes and recorded for 24 hr. Significant genetic differences were found in total sleep, total paradoxical sleep, the number of short sleep episodes, the number of long sleep episodes and the diurnal ratio of total sleep, i.e., some parent strains reliably produced increases in these parameters in hybrids, whereas others produced decreases. Generally, these sleep indices were uncorrelated with, each other. No evidence was found for sex-linkage or early maternal environmental effects on sleep. Paradoxical sleep, the number of long and short sleep episodes and the diurnal ratio of total sleep showed overdominance, i.e., hybrid amounts exceeded those of both parents, suggesting that the overall fitness of the mouse in its wild base population was augmented by a selection for more total sleep, less fragmentation, more even distribution of sleep and less paradoxical sleep. Sleep
Genetics
Diallel
Mice
Paradoxical sleep
RELIABLE differences in sleep patterns have been documented both among individual humans [18] and among various other species, e.g., primitive mammals [ 17], hawks and falcons [13], cats [15], pigs [14], and primates [2]. Sleep patterns have been found largely resistant to environmental manipulations such as prolonged periods of restraint, activity, or partial sleep deprivation [20]. These results, plus the familial histories of sleep-related pathologies such as narcolepsy, enuresis, and somnabulism [1], point to the probable genetic determinance of the sleep process. A pilot study which showed different sleep patterns in two inbred strains [7] supported this position, and so a broader genetic analysis of sleep parameters in the mouse was undertaken. An alternative to the classical Mendelian genetic analysis is the diallel design [3 ], a method suitable for polygenically determined quantitative variables such as are usually the domain of psychologists. Behavioral characteristics are ordinarily of the continuously varying kind and historically have not been amenable to discrete packaging. There has not been much success in ascribing behavior to the effects of single genes, although Mendelian single gene concepts are the basis of polygenic theorizing. The diallel method of behavior genetic analysis provides an effective " 'search' technique for assessing the p r e s e n c e . . , and type of gene action" [ 12]. This involves using several parent strains of inbred animals and all possible first generation hybrids. A
comparison of the hybrid crosses with their respective hybrid reciprocal crosses, i.e., (A female x B male) and its reciprocal mating (A male × B f e m a l e ) a l l o w s inference concerning early maternal environmental effects. The diallel cross method also permits inference about genetic contribution to traits, which has traditionally been partitioned into the following three components: (1) additive variance, which is the sum of the average effects of all genes affecting a trait; (2) dominance variance, which is assumed to be the interaction effects between alleles on homologous chromosomes; (3) epistatic variance, the remainder of non-additive variance which is caused by nonallelic between-chromosome interactions. Other possible data which can be derived from the diallel design are the combining abilities of the strains involved, their general and specific contributions to the hybrid phenotype. The average of a strain's measurements in all its hybrid combinations is its general combining ability. Those specific cases where the hybrid values deviate from the expected mean values of the strains involved reflect its specific combining ability. Combining abilities analyses, therefore, make it possible to assess simultaneously several pure strains gametes' influence in both dams and sires [9]. The single most weighty advantage of the diallel cross method is the generality of results. Both Fuller [8] and Collins [5 ] have noted that using one or two crosses limits conclusions to the specific strains studied. However, a
1Based on author's doctoral dissertation at the University of Florida under the direction of Wilse B. Webb 2Present address: Department of Psychiatry and Human Behavior, University of California, lrvine, California 92664 169
170
FRIEDMANN
diallel matrix derived from several inbred strains delineates the variables of a more generalized population and, therefore, permits greater generalization of conclusions. The possibility of mistakenly generalizing from one of two strains whose characteristics might represent extremes of the population is thereby limited.
METHOD
In the present study five 7 0 - 1 1 0 day old male mice in each of the four original strians, BALB/cJ, C57BL/6J, A/J, and AKR/J, and in the 12 hybrid groups were obtained from Jackson Laboratories, Bar Harbor, Maine. Females were excluded since their sleep patterns vary with the estrus cycle [6]. Surgical implantation was performed under sodium pentobarbital (Nembutal) anesthesia; the dosage was 50 mg/kg, -+0.05 cc, diluted 1"1 with sterile water, and adjusted by trial and error to each genotype since sensitivity varied greatly among the groups. Each mouse was implanted with two stainless steel screws placed unilaterally over frontal and parietal cortex, a bipolar-teflon coated stainless steel electrode in the contralateral dorsal hippocampus, and two nylon insulated wires with 1/16 in. stainless steel pads subcutaneously placed over the nuchal muscles. The electrodes terminated in two miniature pedestals which were chronically attached to the skull with acrylic and two support screws. To determine whether the two pedestals were an encumbrance to the 25 g mice, which might have altered their basic rest/activity cycle, 6 hr of minute by minute visual observations were made of 6 animals before electrode implantation. There were no significant discrepancies in the sleep totals before and after implantation. The animals were maintained on a 12 hr 1300
light-dark cycle, habituated for at least three days to the ventilated, sound-attenuating recording chamber, and for at least 5 hr to connecting leads, before being monitored by electroencephalogram and electromyogram on a Grass Model III EEG at a paper speed of 15 mm/sec for a continuous 24 hr session. The records were scored in 1 min epochs as wakefulness, slow wave sleep, or paradoxical sleep according to the criteria used by Van Twyver [16]. The dependent variables were: (1) TS, the total amount of sleep, both slow wave and paradoxical; (2) PS, the total amount of paradoxical sleep; (3) the total number of sleep episodes of 1 - 3 min duration; (4) the total number of sleep episodes of 4 - 6 min duration; (5) the total number of sleep episodes of 7 - 1 5 min duration; (6) the total number of sleep episodes greater than 15 min duration; (7) DR, the diurnal ratio of total sleep in the light hours to that in the dark. These were analyzed separately for the first and second half of the 24 hr. A three-way, fixed effects analysis of variance [ 11 ] was used to assess dam and sire gametes' effects in both the light and dark phases of the circadian cycle. Although no breeding was done at the University of Florida, the Jackson Laboratories did supply the pedigree of each animal, permitting the dams x sires analysis to be carried out. Grifring's combining abilities analysis of variance [9 ] was used to determine the effect of the four pure strains' gametes on sleep simultaneously in dams and sires, which can be indicative of the general and specific combining abilities of a strain. RESULTS The TS varied greatly among the represented genotypes as illustrated in Fig. 1, which compares each hybrid group
-
1200 L W w
0 0 I,-
0) -
0 0
I
t~ Lu I,-t
0
1100
1000
z
:£ z LU
:£
900
m
n 12
n 21
| 13
I 31
i 14
I 41
i 23
I 32
I 24
I 42
I 34
I 43
HYBRIDS
FIG. 1. Mean minutes total sleep compared in parents and hybrids. The circle respresents a hybrid group, with the endpoints of the line, its parents' strains. (1 = BALB/cJ, 2 = C57BL/6J, 3 = A/J, 4 = AKR/J, e.g., 12 = BALB/cJ dam X C57BL/6J sire.)
GENETIC UNDERPINNINGS OF MOUSE SLEEP
171
mean with its respectivr parent strains' means. When TS is expressed as a percentage of total recording time, there is a 22 per cent range across the 16 groups. The original inbred strains varied in TS time from 9 1 9 - 1 2 3 6 min. Not only does TS vary with each genotype, it is indicated that the strains of the parents do influence the amount of TS in the hybrids. Crosses with an A/J parent showed an average of 26 more min TS than the mean for all the hybrids, while crosses involving the BALB/cJ strain averaged 24 min below the hybrid mean. Additive variance is defined as the average of a strain's measurements in all its hybrid combinations and is a measure of the average effects of the genes acting on a trait. The general combining ability (GCA) component of the second analysis allows inference about the presence of additive genetic variance. By the significance level (p<0.01) of the GCA, as well as dams (p<0.05) and sires (p<0.01) effects (Table 1) it is clear that additive gene action acts on TS. The individual cases where the hybrid values deviate from the expected mean value of the two parent strains involved, or in other words, deviate from that expected on the basis of GCA, reflect specific combining ability (SCA). The SCA effect coupled with the significant dams × sires interaction, both at the 0.01 significance level, indicate that there are specific hybrid groups whose sleep is not the result of simple averaging of parental genes, but is rather a more complex genetic interaction. It is probably
the BALB/cJ x A/J hybrids who are the basis for this result, as it can be seen (Fig. 1) that their circles deviate most from the line midpoints, or the expected midparent values. In order to describe the manner in which genetic influence might be effected, the mean of each hybrid can be compared with the average of its respective parents' means, which is termed the midparent value. If the hybrid deviates from the midparent but does not exceed either parent, the mode of inheritance is termed partial dominance; if the hybrid is equal to one parent, transmission is dominant; it the hybrid exceeds either parent, it is overdominant. Even though the measure of this hybrid deviation from the midparent, the dominance deviation [10], was not significant for TS, in 8 out of 12 cases the hybrid amount exceeded the midparent amount, indicating a trend toward dominant transmission of TS time or partial dominance. Results of PS nearly mirrored those on TS. As shown in Fig. 2, PS totals varied greatly among the genotypes. When PS is viewed as a percentage of TS, there is an 11.8 percent range in PS/TS times across the sixteen genotypes, and specifically in the inbred strains ranges from 11-20%. The significant dams (p<0.025), sires (p<0.01), and GCA effects (p<0.01) indicate that additive genetic variation is present in PS amounts. Crosses with an A/J parent showed an average of 18 min less PS sleep, and those involving a
200
I I 180 L IM MJ ,,d
160 O X 0 a
9t
O
140
II
a_
."
ql
0
o
120
o
Z
0
z
100
0 0
I 12
I 21
I 13
I 31
I 14
I 41
I 23
i 32
i 24
I 42
i 34
! 43
HYIIRIDS
FIG. 2. Mean minutes paradoxical sleep compared in parents and F 1 hybrids. The circle represents a hybrid group, with endpoints of the line, its parents' strains. (1 = BALB/cJ, 2 = C57BL/6J, 3 = A/J, 4 = AKR/J, e.g., 12 = BALB/cJ dam X C57BL/6J sire.)
172
FRIEDMANN TABLE 1 SUMMARY OF ANALYSES OF VARIANCE OF THE SLEEP PARAMETERS, TS, PS, NUMBER OF TS EPISODES OF VARYING LENGTHS, AND DR VARIABLES
Source
df
TS Mean Square
F-Ratio
PS Mean Square
F-Ratio
1-3 min Mean Square F-Ratio
Dams
3
10,461
3.11 *
1,901
3.541-
0.0910
1.26
Sires
3
20,948
6.225
2,906
5.415
2.285
3.17"
Time
1
106,450
65.255
22,681
86.135
Dams X Sires
9
17,415
5.175
1,874
3.48~
12.49 0.7727
26.835 1.00
Dams X Time
3
2,969
1.82
64
0.24
0.4538
0.99
Sires X Time
3
1,031
0.63
564
2.14
0.1891
0.41
Dams X Sires X Time
9
10,468
4.58:~
1.015
2.18"
Error (D, S, D×S)
64
3,367
537
0.721
Error (D×T, SXT, D× S×T)
64
1,632
263
0.466
3
26,938
8.00:~
4,596
SCA
6
20,967
6.23 $
2,707
5.04 $
1.274
1.77
Reciprocals
6
7,393
2.19
209
0.39
0.460
0.64
Early Environment
3
4,228
1.26
218
0.46
0.803
0.11
Dominance Deviation
1
11,603
3.45
7,379
4.50
6.24 t
64
3,367
GCA
Error
*p<0.05
-~p<0.025
6.42:~
1,206
537
8.55 $
13.735
1.076
1.49
0.721
:l:p<0.01
C57BL/6J parent averaged 14 rain more PS than the mean of all the hybrids. The significance of the dams x sires interaction and the SCA effects is probably due to the BALB/cJ x AKR / J crosses whose PS amounts differ from that predicted as a result of additive genetic effects and was, therefore, an interaction effect. When comparing hybrid PS totals with their respective parents' values in Fig. 2, it is clear why the DD was significant (p<0.01). In 7 out of 12 crosses PS was inherited in an overdominant mode and in all but one of the remaining cases was transmitted in at least a partially dominant mode. It is important to note the negative direction of the dominance, which shows PS amounts decreasing in all hybrids beyond that expected on the basis of predicted additive effects of the parental genes. A square root transformation was applied to the sleep episode data in order to achieve homogeneity of within genotype variances. From the analyses of the number of sleep episodes of varying lengths it can be seen (Table 1) that there are no significant dams, sires, or GCA main effects, except the sires effects in the 1 - 3 min category, indicating a general absence of additive genetic influence from the parent strains in hybrids' sleep episode length. SCA was a significant factor in episodes 4 - 6 (p<0.05), and 15+ min in length (p<0.01) as was the dams x sires interaction in the 15+ category (p<0.025). These results indicate a prob-
able dominance (due to allelic interaction) and/or epistatic (nonallelic interaction) effect which was substantiated by the consistent significance of the dominance deviation in all the episode lengths. The A K R/ J x C57BL/6J crosses were probably the ones with the episode lengths outside that predicted by their parental amounts. The inconsistency of the significance levels for varying episode lengths which is evident in Table 1 was probably a reflection of the small n's in the various episode categories. For illustrative purposes, sleep episodes were recategorized into those less than 15 rain and those greater than 15 min in length. Among sleep episodes shorter than 15 min the mode of inheritance in 8 out of 12 cases was over dominant and in 4 cases was partially dominant, all in a negative direction. There are fewer short sleep episodes and more long sleep episodes in hybrid progeny. Considering those sleep episodes more than 15 min long, 11 out of 12 were over dominant, one was partially dominant. All cases were dominant in a positive direction. Interestingly, long sleep episodes of hybrids increase in number significantly above their pure strain parents. Time of day (T) (Table 1) effected every sleep parameter. The significance of the dams x sires x T interactions indicates that the dams x sires interactions are different depending on the time of day and are probably a result of the fact that day sleep is qualitatively different from night
173
GENETIC UNDERPINNINGS OF MOUSE SLEEP TABLE 1 (Contd.)
4 - 6 min Mean Square F-Ratio
0.8797 0.8283
7--15 min Mean Square F-Ratio
1.44
0.9153
1.48
15+ min Mean Square F-Ratio
0.0807
0.0355
2.11
0.0275
1.64
0.1316
7.845
1.35
0.4947
0.80
0.1806
1.39
7.446
15.605
0.6866
6.88t
1.082
1.77
0.5057
0.82
0.3389
2.61t
0.1787
0.31
1.224
2.57
0.1018
1.02
2.027 0.8881
3.53t 1.55
0.9173 0.4804
1.92 1.01
0.4603 0.4070
4.615 4.085
0.612
0.619
0.130
0.574
0.477
0.099
0.0168
1.603
2.62
0.42
0.68
0.06
0.461
1.558
2.55*
0.689
1.11
0.476
0.117
0.19
0.563
0.91
0.133
0.0866
0.14
0.993
1.60
0.2
4.23"
1.81
4.92 0.612
8.045
2.62 0.619
sleep. Webb and Friedmann [18] found that there was a higher probability of PS in rats during the light period than the dark period of the circadian cycle, even with the length of preceding slow wave sleep held constant. This diurnal difference which ranged from 1.07-1.59 in the inbreds, now appears also to be genetically transmitted. On DR there were no dams or sires and, or course, no T main effects. However, there are additive genetic effects (GCA p<0.01), as well as differences beyond those predicted by these additive effects (dams x sires and SCA p< 0.01). Figure 3 illustrates the p< 0.01 significance level of DD, 8 out of 12 hybrids were negatively over dominant and of those remaining, 3 were partially negatively dominant. The ratio of day to night sleep is influenced by varying genotypes and appears to decrease in the hybrids. No reciprocals' effect was found on any of the variables, that is, the sleep of hybrids in cell 12 was not found to differ from that of cell 21 in any way, in addition to which there were no significant early maternal environmental effects on the seven variables to indicate the presence of sex-linkage or any basic perinatal differences between the strains. Had the groups above and below the diagonal diverged, it would have been necessary to postulate early maternal effects acting on sleep, assuming that the genotypes are the same; or alternately, that there is possible residual genetic variance and/or that part of the X chromosome missing in the Y is not necessarily identically, present across reciprocal males.
0.130
F-Ratio
0.62
18.945
10.87
DR Mean Square
0.0833
4.965
3.675
0.1532
9.115
1.03
0.0341
2.03
1.54
0.04
2.38
13.965
1.23
73.335
0.0168
It is important to know if these genetically influenced sleep parameters can vary independently of each other, therefore, correlation coefficients were computed between the sleep parameters within each genotype. The hybrids and their reciprocals were combined giving a N = 10 for each genotype except the four pure strains where N = 5. The only emerging consistent patterns of covariance in spite of the small N are that as TS increases, the DR decreases, and TS and PS appear both negatively correlated with the short sleep episodes. The indication is that, with the exception of these three possible correlations, there appear to be no axes along which sleep parameters covary in a consistent fashion. DISCUSSION
It is clear that the various sleep parameters are largely genetically determined. TS, PS, and DR are influenced in an additive fashion by the genotypes of the parents. For each of these variables, there is one parent strain which predictably increases and another which decreases these hybrid sleep parameters. Superimposed on the general additivity is dominant variation, a result of allelic interaction, which causes some hybrids to differ from that predictable on the basis of additivity alone. The remaining variable, sleep episode length, shows no evidence of additive genetic determinance but is transmitted in a wholly dominant fashion. The implications of this dominant genetic influence on
FRIEDMANN
174
1.50
-
1-40
L Ma MI
1.30
li
.,.a
4(
k,-
0 1.20 0 I--
4( nt
1.10 0 z
0
at,
0
0 0
a
1.00
0
0
0 • 90
_
I
I
i
i
,
i
i
i
l
i
|
I
12
21
13
31
14
41
23
32
24
42
34
43
HYBRIDS
FIG. 3. Diurnal ratio of total day to night sleep compared in parents and F t hybrids. The circle represents a hybrid group, with endpoints of the line, its parents' strains. (1 = BALB/cJ, 2 = C57BL/6J, 3 = A/J, 4 = AKR/J, e.g., 12 = BALB/cJ dam × C57BL/6J sire.) sleep deserves consideration. Total sleep shows only a trend toward dominant transmission whereas every other variable shows overdominant genetic transmission. This hybrid deviation from midparent values is termed heterosis, or hybrid vigor, and has reference to the usual superior viability of the hybrid with respect to its inbred parents. Knowing that the mode of sleep inheritance is generally heterotic, it is important to examine, as Bruell [4] has so thoroughly done, the implications of this mode of inheritance. He ascribes selective advantage to heterotic traits. Dominant traits have selective advantage. Since with each sleep parameter there is an asymmetry of phenotypic expression, that is, the means of sleep patterns in the hybrids deviated from the expected midparent sleep means, it can be inferred that the selection history with respect to sleep was for extreme and not intermediate values. Had the various sleep parameters expressed a symmetrical distribution, the hybrid values equalling the midparent values, at least two explanations could be offered. One explanation would suggest a past selection pressure for an optimum of intermediacy. A study of emotionality of rats [3 ] indicated such a selection for intermediacy. Highly emotional or extremely passive animals might be expected to make inappropriate responses in food-getting and mating situations, thereby limiting their survival and procreation potential. The emotionally inter-
mediate animal would have a higher probability of success and as a result, intermediate emotionality would be selected for. An alternative explanation for intermediate expression of traits would posit a gene pool on the whole untouched by selection pressure in the current environment. Such a pool is maintained through chance (mutation) and, more importantly, through pleiotropy. Pleiotropy is the case where a gene effects more than one trait. Mutations and pleiotropy, plus the fact that genes are selected for on the basis of the overall fitness of the organism, result in a random gene pool made up of dominant positive, dominant negative and additive genes, and thus a truly random, symmetrical phenotypic distribution is generated [4]. An example of such a lack of past selection pressure is alcohol preference in mice [8 ] which is genetically transmitted and exhibits hybrid intermediacy. However, there being no alcohol in the mouse's natural environment this is probably a pleiotropic or neutral effect, not one selected for. From the symmetry, or lack of it, found in the distribution of hybrid values, it is possible to theorize about the history of selection pressure exerted on a trait such as total amount of sleep. The asymmetrical distribution of TS indicates a past selection for longer sleepers. From here it is but a short step to the suggestion that traits which are selected for in the extreme, as opposed to the intermediate range,
GENETIC UNDERPINNINGS OF MOUSE SLEEP
175
c o n t r i b u t e to the general fitness of the organism. With i n t e r m e d i a t e range characters, a history of selective advantage or neutrality is possible. However, since there is no i n t e r m e d i a c y in the distribution of any hybrid sleep param-
eter it is possible to h y p o t h e s i z e that more total sleep, less fragmentation, m o r e even distribution o f sleep, and decreased paradoxical sleep c o n t r i b u t e d to the fitness of the mouse in its wild base population.
REFERENCES 1. Altschuler, K. Z. and D. R. Brebbia. Potential contributions of genetics in sleep research. Psychosom. Med. 134: 103-109, 1966. 2. Bert, J., D. F. Kripke and J. Rhodes. Electroencephalogram of the mature chimpanzee: twenty-four recordings. Electroenceph, clin. Neurophysiol. 28: 368-373, 1970. 3. Broadburst, P. L. Experiments in psychogenetics. In: Experience in Personality. Vol. 1. Psychogenetics and Psychopharmacology, edited by H. J. Eysenck. London: Routledge, pp. 1-102, 1960. 4. Bruell, J. H. Evolution and heterosis. In: Behavior-genetic Analysis, edited by J. Hirsch. New York: McGraw-Hill, 1967, pp. 421-438. 5. Collins, R. L. Inheritance of avoidance conditioning in mice: a diallel study. Science 143:1188-1190, 1964. 6. Colvin, G. B., D. I. Whitmoyer, R. D. Lisk, D. O. Waiter and C. H. Sawyer. Changes in sleep-wakefulness in female rats during circadian and estrous cycles. Brain Res. 7: 173-181, 1968. 7. Friedmann, J. K. and W. B. Webb. Genetics determinants of the sleep response. Psychophysiology 7: 340, 1970. 8. Fuller, J. L. Measurement of alcohol preference in genetic experiments. J. comp. physiol. Psychol. 57: 85-88, 1964. 9. Griffing, B. Concept of general and specific combining ability in relation to diallel crossing system. Aust. J. biol. Scl. 9: 463-493, 1956.
10. Hayman, B. I. The analysis of variance in diallel tables. Biometrics 39: 235-244, 1954. 11. Kirk, R. E. Experimental Design Procedures for the Behavioral Sciences. Belmont, Calif.: Brooks/Cole, 1968. 12. Newell, T. G. Three biometrical genetic analyses of activity in the mouse. J. comp. physiol. Psychol. 70: 37-47, 1969. 13. Rojas-Ramirez, J. and E. Tauber. Paradoxical sleep in two species of avian predator (Falconiformes). Science 167: 1754-1755, 1970. 14. Ruckebusch, Y. and M. T. Morel. Etude polygraphique du sommeil chez le porc. C. R. Soc. Biol. 162: 1346-1354, 1968. 15. Sterman, M. B., T. Knauss, D. Lehmann and C. D. Clemente. Circadian sleep and waking patterns in the laboratory cat. Electroenceph. clin. Neurophysiol. 19: 509-513, 1965. 16. Van Twyver, H. Sleep patterns of five rodent species. Physiol. Behav. 4: 901-905, 1969. 17. Van Twyver, H. B. and T. Allison. Sleep in the opossum (Didelphis marsupialis}. Electroenceph. clin. Neurophysiol. 29: 181-186, 1970. 18. Webb, W. B. Individual differences in sleep length. In: Sleep and Dreaming, edited by E. Hartmann. Boston: Little Brown, 1970, pp. 44-47. 19. Webb, W. B. and J. K. Friedmann. Some temporal characteristics of paradoxical (LVF) sleep occurrence in the rat. Electro. enceph, clin. Neurophysiol. 30: 453-456, 1971. 20. Webb, W. B. and J. K. Friedmann. Attempts to modify the sleep patterns of the rat. Physiol. Behav. 6: 459-460, 1971.
A Diallel Analysis of the Genetic Underpinnings of Mouse S l e e p I JOYCE K. FRIEDMANN 2
University o f Florida, Gainesville, Florida, U.S.A.
(Received 15 March 1972) FRIEDMANN, J. K. A Diallel analysis of the genetic underpinnings of mouse sleep. PHYSIOL. BEHAV. 12(2) 169-175, 1974.-Four highly inbred mouse strains were crossbred in all possible combinations. Five young adult males in each of the four original and twelve offspring groups were chronically implanted with cortical, hippocampal and nuchal electrodes and recorded for 24 hr. Significant genetic differences were found in total sleep, total paradoxical sleep, the number of short sleep episodes, the number of long sleep episodes and the diurnal ratio of total sleep, i.e., some parent strains reliably produced increases in these parameters in hybrids, whereas others produced decreases. Generally, these sleep indices were uncorrelated with, each other. No evidence was found for sex-linkage or early maternal environmental effects on sleep. Paradoxical sleep, the number of long and short sleep episodes and the diurnal ratio of total sleep showed overdominance, i.e., hybrid amounts exceeded those of both parents, suggesting that the overall fitness of the mouse in its wild base population was augmented by a selection for more total sleep, less fragmentation, more even distribution of sleep and less paradoxical sleep. Sleep
Genetics
Diallel
Mice
Paradoxical sleep
RELIABLE differences in sleep patterns have been documented both among individual humans [18] and among various other species, e.g., primitive mammals [ 17], hawks and falcons [13], cats [15], pigs [14], and primates [2]. Sleep patterns have been found largely resistant to environmental manipulations such as prolonged periods of restraint, activity, or partial sleep deprivation [20]. These results, plus the familial histories of sleep-related pathologies such as narcolepsy, enuresis, and somnabulism [1], point to the probable genetic determinance of the sleep process. A pilot study which showed different sleep patterns in two inbred strains [7] supported this position, and so a broader genetic analysis of sleep parameters in the mouse was undertaken. An alternative to the classical Mendelian genetic analysis is the diallel design [3 ], a method suitable for polygenically determined quantitative variables such as are usually the domain of psychologists. Behavioral characteristics are ordinarily of the continuously varying kind and historically have not been amenable to discrete packaging. There has not been much success in ascribing behavior to the effects of single genes, although Mendelian single gene concepts are the basis of polygenic theorizing. The diallel method of behavior genetic analysis provides an effective " 'search' technique for assessing the p r e s e n c e . . , and type of gene action" [ 12]. This involves using several parent strains of inbred animals and all possible first generation hybrids. A
comparison of the hybrid crosses with their respective hybrid reciprocal crosses, i.e., (A female x B male) and its reciprocal mating (A male × B f e m a l e ) a l l o w s inference concerning early maternal environmental effects. The diallel cross method also permits inference about genetic contribution to traits, which has traditionally been partitioned into the following three components: (1) additive variance, which is the sum of the average effects of all genes affecting a trait; (2) dominance variance, which is assumed to be the interaction effects between alleles on homologous chromosomes; (3) epistatic variance, the remainder of non-additive variance which is caused by nonallelic between-chromosome interactions. Other possible data which can be derived from the diallel design are the combining abilities of the strains involved, their general and specific contributions to the hybrid phenotype. The average of a strain's measurements in all its hybrid combinations is its general combining ability. Those specific cases where the hybrid values deviate from the expected mean values of the strains involved reflect its specific combining ability. Combining abilities analyses, therefore, make it possible to assess simultaneously several pure strains gametes' influence in both dams and sires [9]. The single most weighty advantage of the diallel cross method is the generality of results. Both Fuller [8] and Collins [5 ] have noted that using one or two crosses limits conclusions to the specific strains studied. However, a
1Based on author's doctoral dissertation at the University of Florida under the direction of Wilse B. Webb 2Present address: Department of Psychiatry and Human Behavior, University of California, lrvine, California 92664 169
170
FRIEDMANN
diallel matrix derived from several inbred strains delineates the variables of a more generalized population and, therefore, permits greater generalization of conclusions. The possibility of mistakenly generalizing from one of two strains whose characteristics might represent extremes of the population is thereby limited.
METHOD
In the present study five 7 0 - 1 1 0 day old male mice in each of the four original strians, BALB/cJ, C57BL/6J, A/J, and AKR/J, and in the 12 hybrid groups were obtained from Jackson Laboratories, Bar Harbor, Maine. Females were excluded since their sleep patterns vary with the estrus cycle [6]. Surgical implantation was performed under sodium pentobarbital (Nembutal) anesthesia; the dosage was 50 mg/kg, -+0.05 cc, diluted 1"1 with sterile water, and adjusted by trial and error to each genotype since sensitivity varied greatly among the groups. Each mouse was implanted with two stainless steel screws placed unilaterally over frontal and parietal cortex, a bipolar-teflon coated stainless steel electrode in the contralateral dorsal hippocampus, and two nylon insulated wires with 1/16 in. stainless steel pads subcutaneously placed over the nuchal muscles. The electrodes terminated in two miniature pedestals which were chronically attached to the skull with acrylic and two support screws. To determine whether the two pedestals were an encumbrance to the 25 g mice, which might have altered their basic rest/activity cycle, 6 hr of minute by minute visual observations were made of 6 animals before electrode implantation. There were no significant discrepancies in the sleep totals before and after implantation. The animals were maintained on a 12 hr 1300
light-dark cycle, habituated for at least three days to the ventilated, sound-attenuating recording chamber, and for at least 5 hr to connecting leads, before being monitored by electroencephalogram and electromyogram on a Grass Model III EEG at a paper speed of 15 mm/sec for a continuous 24 hr session. The records were scored in 1 min epochs as wakefulness, slow wave sleep, or paradoxical sleep according to the criteria used by Van Twyver [16]. The dependent variables were: (1) TS, the total amount of sleep, both slow wave and paradoxical; (2) PS, the total amount of paradoxical sleep; (3) the total number of sleep episodes of 1 - 3 min duration; (4) the total number of sleep episodes of 4 - 6 min duration; (5) the total number of sleep episodes of 7 - 1 5 min duration; (6) the total number of sleep episodes greater than 15 min duration; (7) DR, the diurnal ratio of total sleep in the light hours to that in the dark. These were analyzed separately for the first and second half of the 24 hr. A three-way, fixed effects analysis of variance [ 11 ] was used to assess dam and sire gametes' effects in both the light and dark phases of the circadian cycle. Although no breeding was done at the University of Florida, the Jackson Laboratories did supply the pedigree of each animal, permitting the dams x sires analysis to be carried out. Grifring's combining abilities analysis of variance [9 ] was used to determine the effect of the four pure strains' gametes on sleep simultaneously in dams and sires, which can be indicative of the general and specific combining abilities of a strain. RESULTS The TS varied greatly among the represented genotypes as illustrated in Fig. 1, which compares each hybrid group
-
1200 L W w
0 0 I,-
0) -
0 0
I
t~ Lu I,-t
0
1100
1000
z
:£ z LU
:£
900
m
n 12
n 21
| 13
I 31
i 14
I 41
i 23
I 32
I 24
I 42
I 34
I 43
HYBRIDS
FIG. 1. Mean minutes total sleep compared in parents and hybrids. The circle respresents a hybrid group, with the endpoints of the line, its parents' strains. (1 = BALB/cJ, 2 = C57BL/6J, 3 = A/J, 4 = AKR/J, e.g., 12 = BALB/cJ dam X C57BL/6J sire.)
GENETIC UNDERPINNINGS OF MOUSE SLEEP
171
mean with its respectivr parent strains' means. When TS is expressed as a percentage of total recording time, there is a 22 per cent range across the 16 groups. The original inbred strains varied in TS time from 9 1 9 - 1 2 3 6 min. Not only does TS vary with each genotype, it is indicated that the strains of the parents do influence the amount of TS in the hybrids. Crosses with an A/J parent showed an average of 26 more min TS than the mean for all the hybrids, while crosses involving the BALB/cJ strain averaged 24 min below the hybrid mean. Additive variance is defined as the average of a strain's measurements in all its hybrid combinations and is a measure of the average effects of the genes acting on a trait. The general combining ability (GCA) component of the second analysis allows inference about the presence of additive genetic variance. By the significance level (p<0.01) of the GCA, as well as dams (p<0.05) and sires (p<0.01) effects (Table 1) it is clear that additive gene action acts on TS. The individual cases where the hybrid values deviate from the expected mean value of the two parent strains involved, or in other words, deviate from that expected on the basis of GCA, reflect specific combining ability (SCA). The SCA effect coupled with the significant dams × sires interaction, both at the 0.01 significance level, indicate that there are specific hybrid groups whose sleep is not the result of simple averaging of parental genes, but is rather a more complex genetic interaction. It is probably
the BALB/cJ x A/J hybrids who are the basis for this result, as it can be seen (Fig. 1) that their circles deviate most from the line midpoints, or the expected midparent values. In order to describe the manner in which genetic influence might be effected, the mean of each hybrid can be compared with the average of its respective parents' means, which is termed the midparent value. If the hybrid deviates from the midparent but does not exceed either parent, the mode of inheritance is termed partial dominance; if the hybrid is equal to one parent, transmission is dominant; it the hybrid exceeds either parent, it is overdominant. Even though the measure of this hybrid deviation from the midparent, the dominance deviation [10], was not significant for TS, in 8 out of 12 cases the hybrid amount exceeded the midparent amount, indicating a trend toward dominant transmission of TS time or partial dominance. Results of PS nearly mirrored those on TS. As shown in Fig. 2, PS totals varied greatly among the genotypes. When PS is viewed as a percentage of TS, there is an 11.8 percent range in PS/TS times across the sixteen genotypes, and specifically in the inbred strains ranges from 11-20%. The significant dams (p<0.025), sires (p<0.01), and GCA effects (p<0.01) indicate that additive genetic variation is present in PS amounts. Crosses with an A/J parent showed an average of 18 min less PS sleep, and those involving a
200
I I 180 L IM MJ ,,d
160 O X 0 a
9t
O
140
II
a_
."
ql
0
o
120
o
Z
0
z
100
0 0
I 12
I 21
I 13
I 31
I 14
I 41
I 23
i 32
i 24
I 42
i 34
! 43
HYIIRIDS
FIG. 2. Mean minutes paradoxical sleep compared in parents and F 1 hybrids. The circle represents a hybrid group, with endpoints of the line, its parents' strains. (1 = BALB/cJ, 2 = C57BL/6J, 3 = A/J, 4 = AKR/J, e.g., 12 = BALB/cJ dam X C57BL/6J sire.)
172
FRIEDMANN TABLE 1 SUMMARY OF ANALYSES OF VARIANCE OF THE SLEEP PARAMETERS, TS, PS, NUMBER OF TS EPISODES OF VARYING LENGTHS, AND DR VARIABLES
Source
df
TS Mean Square
F-Ratio
PS Mean Square
F-Ratio
1-3 min Mean Square F-Ratio
Dams
3
10,461
3.11 *
1,901
3.541-
0.0910
1.26
Sires
3
20,948
6.225
2,906
5.415
2.285
3.17"
Time
1
106,450
65.255
22,681
86.135
Dams X Sires
9
17,415
5.175
1,874
3.48~
12.49 0.7727
26.835 1.00
Dams X Time
3
2,969
1.82
64
0.24
0.4538
0.99
Sires X Time
3
1,031
0.63
564
2.14
0.1891
0.41
Dams X Sires X Time
9
10,468
4.58:~
1.015
2.18"
Error (D, S, D×S)
64
3,367
537
0.721
Error (D×T, SXT, D× S×T)
64
1,632
263
0.466
3
26,938
8.00:~
4,596
SCA
6
20,967
6.23 $
2,707
5.04 $
1.274
1.77
Reciprocals
6
7,393
2.19
209
0.39
0.460
0.64
Early Environment
3
4,228
1.26
218
0.46
0.803
0.11
Dominance Deviation
1
11,603
3.45
7,379
4.50
6.24 t
64
3,367
GCA
Error
*p<0.05
-~p<0.025
6.42:~
1,206
537
8.55 $
13.735
1.076
1.49
0.721
:l:p<0.01
C57BL/6J parent averaged 14 rain more PS than the mean of all the hybrids. The significance of the dams x sires interaction and the SCA effects is probably due to the BALB/cJ x AKR / J crosses whose PS amounts differ from that predicted as a result of additive genetic effects and was, therefore, an interaction effect. When comparing hybrid PS totals with their respective parents' values in Fig. 2, it is clear why the DD was significant (p<0.01). In 7 out of 12 crosses PS was inherited in an overdominant mode and in all but one of the remaining cases was transmitted in at least a partially dominant mode. It is important to note the negative direction of the dominance, which shows PS amounts decreasing in all hybrids beyond that expected on the basis of predicted additive effects of the parental genes. A square root transformation was applied to the sleep episode data in order to achieve homogeneity of within genotype variances. From the analyses of the number of sleep episodes of varying lengths it can be seen (Table 1) that there are no significant dams, sires, or GCA main effects, except the sires effects in the 1 - 3 min category, indicating a general absence of additive genetic influence from the parent strains in hybrids' sleep episode length. SCA was a significant factor in episodes 4 - 6 (p<0.05), and 15+ min in length (p<0.01) as was the dams x sires interaction in the 15+ category (p<0.025). These results indicate a prob-
able dominance (due to allelic interaction) and/or epistatic (nonallelic interaction) effect which was substantiated by the consistent significance of the dominance deviation in all the episode lengths. The A K R/ J x C57BL/6J crosses were probably the ones with the episode lengths outside that predicted by their parental amounts. The inconsistency of the significance levels for varying episode lengths which is evident in Table 1 was probably a reflection of the small n's in the various episode categories. For illustrative purposes, sleep episodes were recategorized into those less than 15 rain and those greater than 15 min in length. Among sleep episodes shorter than 15 min the mode of inheritance in 8 out of 12 cases was over dominant and in 4 cases was partially dominant, all in a negative direction. There are fewer short sleep episodes and more long sleep episodes in hybrid progeny. Considering those sleep episodes more than 15 min long, 11 out of 12 were over dominant, one was partially dominant. All cases were dominant in a positive direction. Interestingly, long sleep episodes of hybrids increase in number significantly above their pure strain parents. Time of day (T) (Table 1) effected every sleep parameter. The significance of the dams x sires x T interactions indicates that the dams x sires interactions are different depending on the time of day and are probably a result of the fact that day sleep is qualitatively different from night
173
GENETIC UNDERPINNINGS OF MOUSE SLEEP TABLE 1 (Contd.)
4 - 6 min Mean Square F-Ratio
0.8797 0.8283
7--15 min Mean Square F-Ratio
1.44
0.9153
1.48
15+ min Mean Square F-Ratio
0.0807
0.0355
2.11
0.0275
1.64
0.1316
7.845
1.35
0.4947
0.80
0.1806
1.39
7.446
15.605
0.6866
6.88t
1.082
1.77
0.5057
0.82
0.3389
2.61t
0.1787
0.31
1.224
2.57
0.1018
1.02
2.027 0.8881
3.53t 1.55
0.9173 0.4804
1.92 1.01
0.4603 0.4070
4.615 4.085
0.612
0.619
0.130
0.574
0.477
0.099
0.0168
1.603
2.62
0.42
0.68
0.06
0.461
1.558
2.55*
0.689
1.11
0.476
0.117
0.19
0.563
0.91
0.133
0.0866
0.14
0.993
1.60
0.2
4.23"
1.81
4.92 0.612
8.045
2.62 0.619
sleep. Webb and Friedmann [18] found that there was a higher probability of PS in rats during the light period than the dark period of the circadian cycle, even with the length of preceding slow wave sleep held constant. This diurnal difference which ranged from 1.07-1.59 in the inbreds, now appears also to be genetically transmitted. On DR there were no dams or sires and, or course, no T main effects. However, there are additive genetic effects (GCA p<0.01), as well as differences beyond those predicted by these additive effects (dams x sires and SCA p< 0.01). Figure 3 illustrates the p< 0.01 significance level of DD, 8 out of 12 hybrids were negatively over dominant and of those remaining, 3 were partially negatively dominant. The ratio of day to night sleep is influenced by varying genotypes and appears to decrease in the hybrids. No reciprocals' effect was found on any of the variables, that is, the sleep of hybrids in cell 12 was not found to differ from that of cell 21 in any way, in addition to which there were no significant early maternal environmental effects on the seven variables to indicate the presence of sex-linkage or any basic perinatal differences between the strains. Had the groups above and below the diagonal diverged, it would have been necessary to postulate early maternal effects acting on sleep, assuming that the genotypes are the same; or alternately, that there is possible residual genetic variance and/or that part of the X chromosome missing in the Y is not necessarily identically, present across reciprocal males.
0.130
F-Ratio
0.62
18.945
10.87
DR Mean Square
0.0833
4.965
3.675
0.1532
9.115
1.03
0.0341
2.03
1.54
0.04
2.38
13.965
1.23
73.335
0.0168
It is important to know if these genetically influenced sleep parameters can vary independently of each other, therefore, correlation coefficients were computed between the sleep parameters within each genotype. The hybrids and their reciprocals were combined giving a N = 10 for each genotype except the four pure strains where N = 5. The only emerging consistent patterns of covariance in spite of the small N are that as TS increases, the DR decreases, and TS and PS appear both negatively correlated with the short sleep episodes. The indication is that, with the exception of these three possible correlations, there appear to be no axes along which sleep parameters covary in a consistent fashion. DISCUSSION
It is clear that the various sleep parameters are largely genetically determined. TS, PS, and DR are influenced in an additive fashion by the genotypes of the parents. For each of these variables, there is one parent strain which predictably increases and another which decreases these hybrid sleep parameters. Superimposed on the general additivity is dominant variation, a result of allelic interaction, which causes some hybrids to differ from that predictable on the basis of additivity alone. The remaining variable, sleep episode length, shows no evidence of additive genetic determinance but is transmitted in a wholly dominant fashion. The implications of this dominant genetic influence on
FRIEDMANN
174
1.50
-
1-40
L Ma MI
1.30
li
.,.a
4(
k,-
0 1.20 0 I--
4( nt
1.10 0 z
0
at,
0
0 0
a
1.00
0
0
0 • 90
_
I
I
i
i
,
i
i
i
l
i
|
I
12
21
13
31
14
41
23
32
24
42
34
43
HYBRIDS
FIG. 3. Diurnal ratio of total day to night sleep compared in parents and F t hybrids. The circle represents a hybrid group, with endpoints of the line, its parents' strains. (1 = BALB/cJ, 2 = C57BL/6J, 3 = A/J, 4 = AKR/J, e.g., 12 = BALB/cJ dam × C57BL/6J sire.) sleep deserves consideration. Total sleep shows only a trend toward dominant transmission whereas every other variable shows overdominant genetic transmission. This hybrid deviation from midparent values is termed heterosis, or hybrid vigor, and has reference to the usual superior viability of the hybrid with respect to its inbred parents. Knowing that the mode of sleep inheritance is generally heterotic, it is important to examine, as Bruell [4] has so thoroughly done, the implications of this mode of inheritance. He ascribes selective advantage to heterotic traits. Dominant traits have selective advantage. Since with each sleep parameter there is an asymmetry of phenotypic expression, that is, the means of sleep patterns in the hybrids deviated from the expected midparent sleep means, it can be inferred that the selection history with respect to sleep was for extreme and not intermediate values. Had the various sleep parameters expressed a symmetrical distribution, the hybrid values equalling the midparent values, at least two explanations could be offered. One explanation would suggest a past selection pressure for an optimum of intermediacy. A study of emotionality of rats [3 ] indicated such a selection for intermediacy. Highly emotional or extremely passive animals might be expected to make inappropriate responses in food-getting and mating situations, thereby limiting their survival and procreation potential. The emotionally inter-
mediate animal would have a higher probability of success and as a result, intermediate emotionality would be selected for. An alternative explanation for intermediate expression of traits would posit a gene pool on the whole untouched by selection pressure in the current environment. Such a pool is maintained through chance (mutation) and, more importantly, through pleiotropy. Pleiotropy is the case where a gene effects more than one trait. Mutations and pleiotropy, plus the fact that genes are selected for on the basis of the overall fitness of the organism, result in a random gene pool made up of dominant positive, dominant negative and additive genes, and thus a truly random, symmetrical phenotypic distribution is generated [4]. An example of such a lack of past selection pressure is alcohol preference in mice [8 ] which is genetically transmitted and exhibits hybrid intermediacy. However, there being no alcohol in the mouse's natural environment this is probably a pleiotropic or neutral effect, not one selected for. From the symmetry, or lack of it, found in the distribution of hybrid values, it is possible to theorize about the history of selection pressure exerted on a trait such as total amount of sleep. The asymmetrical distribution of TS indicates a past selection for longer sleepers. From here it is but a short step to the suggestion that traits which are selected for in the extreme, as opposed to the intermediate range,
GENETIC UNDERPINNINGS OF MOUSE SLEEP
175
c o n t r i b u t e to the general fitness of the organism. With i n t e r m e d i a t e range characters, a history of selective advantage or neutrality is possible. However, since there is no i n t e r m e d i a c y in the distribution of any hybrid sleep param-
eter it is possible to h y p o t h e s i z e that more total sleep, less fragmentation, m o r e even distribution o f sleep, and decreased paradoxical sleep c o n t r i b u t e d to the fitness of the mouse in its wild base population.
REFERENCES 1. Altschuler, K. Z. and D. R. Brebbia. Potential contributions of genetics in sleep research. Psychosom. Med. 134: 103-109, 1966. 2. Bert, J., D. F. Kripke and J. Rhodes. Electroencephalogram of the mature chimpanzee: twenty-four recordings. Electroenceph, clin. Neurophysiol. 28: 368-373, 1970. 3. Broadburst, P. L. Experiments in psychogenetics. In: Experience in Personality. Vol. 1. Psychogenetics and Psychopharmacology, edited by H. J. Eysenck. London: Routledge, pp. 1-102, 1960. 4. Bruell, J. H. Evolution and heterosis. In: Behavior-genetic Analysis, edited by J. Hirsch. New York: McGraw-Hill, 1967, pp. 421-438. 5. Collins, R. L. Inheritance of avoidance conditioning in mice: a diallel study. Science 143:1188-1190, 1964. 6. Colvin, G. B., D. I. Whitmoyer, R. D. Lisk, D. O. Waiter and C. H. Sawyer. Changes in sleep-wakefulness in female rats during circadian and estrous cycles. Brain Res. 7: 173-181, 1968. 7. Friedmann, J. K. and W. B. Webb. Genetics determinants of the sleep response. Psychophysiology 7: 340, 1970. 8. Fuller, J. L. Measurement of alcohol preference in genetic experiments. J. comp. physiol. Psychol. 57: 85-88, 1964. 9. Griffing, B. Concept of general and specific combining ability in relation to diallel crossing system. Aust. J. biol. Scl. 9: 463-493, 1956.
10. Hayman, B. I. The analysis of variance in diallel tables. Biometrics 39: 235-244, 1954. 11. Kirk, R. E. Experimental Design Procedures for the Behavioral Sciences. Belmont, Calif.: Brooks/Cole, 1968. 12. Newell, T. G. Three biometrical genetic analyses of activity in the mouse. J. comp. physiol. Psychol. 70: 37-47, 1969. 13. Rojas-Ramirez, J. and E. Tauber. Paradoxical sleep in two species of avian predator (Falconiformes). Science 167: 1754-1755, 1970. 14. Ruckebusch, Y. and M. T. Morel. Etude polygraphique du sommeil chez le porc. C. R. Soc. Biol. 162: 1346-1354, 1968. 15. Sterman, M. B., T. Knauss, D. Lehmann and C. D. Clemente. Circadian sleep and waking patterns in the laboratory cat. Electroenceph. clin. Neurophysiol. 19: 509-513, 1965. 16. Van Twyver, H. Sleep patterns of five rodent species. Physiol. Behav. 4: 901-905, 1969. 17. Van Twyver, H. B. and T. Allison. Sleep in the opossum (Didelphis marsupialis}. Electroenceph. clin. Neurophysiol. 29: 181-186, 1970. 18. Webb, W. B. Individual differences in sleep length. In: Sleep and Dreaming, edited by E. Hartmann. Boston: Little Brown, 1970, pp. 44-47. 19. Webb, W. B. and J. K. Friedmann. Some temporal characteristics of paradoxical (LVF) sleep occurrence in the rat. Electro. enceph, clin. Neurophysiol. 30: 453-456, 1971. 20. Webb, W. B. and J. K. Friedmann. Attempts to modify the sleep patterns of the rat. Physiol. Behav. 6: 459-460, 1971.